research article physicochemical properties and in...

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2013 Article ID 653892 10 pageshttpdxdoiorg1011552013653892

Research ArticlePhysicochemical Properties and In Vitro Cytotoxicity Studies ofChitosan as a Potential Carrier for Dicer-Substrate siRNA

Maria Abdul Ghafoor Raja1 Haliza Katas1 Zariyantey Abd Hamid2 and Nur Atiqah Razali1

1 Centre for Drug Delivery Research Faculty of Pharmacy Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz50300 Kuala Lumpur Malaysia

2 Program of Biomedical Science School of Diagnostic and Applied Health Sciences Faculty of Health SciencesUniversiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz 50300 Kuala Lumpur Malaysia

Correspondence should be addressed to Haliza Katas haliz12hotmailcom

Received 17 May 2013 Revised 26 July 2013 Accepted 27 July 2013

Academic Editor Hongchen Chen Gu

Copyright copy 2013 Maria Abdul Ghafoor Raja et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Recently Dicer-substrate small interfering RNA (DsiRNA) has gained attention owing to its greater potency over small interferingRNA (siRNA) However the use of DsiRNA is restricted by its rapid degradation in vitro To address this issue chitosannanoparticulate deliver yplatform for the Dicer-substrate siRNA (DsiRNA) was developed and characterized Nanoparticles wereprepared by simple complexation and ionic gelation methods The mean particle size of DsiRNA-adsorbed chitosan nanospheres(DsiRNA-CS NPs) prepared by the ionic gelation method ranged from 225 to 335 nm while simple complexation yielded DsiRNA-chitosan complexes (DsiRNA-CS complexes) ranging from 270 to 730 nmThe zeta potential of both types of nanoparticles rangedfrom +40 to +65mV TEM and AFMmicrographs revealed spherical and irregular morphology of DsiRNA-CS NPs and DsiRNA-CS complexes ATR-FTIR spectroscopy confirmed the presence of DsiRNA in the CS NPscomplexes Both types of nanoparticlesexhibited sustained release and high binding and encapsulation (100) efficiency of DsiRNA DsiRNA-CS NPscomplexes showedlow concentration-dependent cytotoxicity in vitro DsiRNA-CS NPs showed better stability than the complexes when stored at 4and 25∘C Thus it is anticipated that CS NPs are promising vectors for DsiRNA delivery due to their stability safety and cost-effectiveness

1 Introduction

Successful gene delivery and expression remain a majordifficulty thatmust be overcome before genetic therapies gainclinical acceptance There are however barriers to effectivedelivery of these molecules as most nucleic acids are quicklydegraded and cleared by nucleases and macrophages There-fore a degree of protection is required for incorporation intotheir delivery system [1] Currently there is much emphasison the further development of nucleic acid delivery systems

Viral and nonviral vectors have been used as gene deliverycarriers Even though viral vectors have higher transfectionefficiencies in most cells safety concerns were raised innumerous clinical trials [2] Nonviral vectors have garneredmuch consideration owing to their ease of synthesis andmodification low immunogenicity and controllable size [3]Positively charged cationic polymers can effectively bind to

and protect nucleic acids such as DNA oligonucleotides andsiRNAs Nonviral delivery systems using cationic liposomesand polymers such as polyethylenimine (PEI) poly(L-lysine)(PLL) and their respective derivatives have been used to con-dense plasmid DNA (pDNA) or siRNA to form nanoparti-cles [4ndash6] However liposomal-based formulations normallyleading to cell toxicity [7] are quickly cleared from the blood-stream [8] Among all the recentmaterials used for polymericnanoparticles synthesis chitosan (CS) a natural plentifulbiopolymer obtained by chitin deacetylation has gained con-siderable interest [9] CS is a linear polysaccharide composedof glucosamine andN-acetyl glucosamine residues and can bederived by partial deacetylation of chitin [10] CS is known tobe biocompatible less toxic nonimmunogenic and degrad-able by enzymes [11ndash13] CS has been used extensively inmany drug delivery applications especially in gene deliverysystems because of its positively charged amines that allow

2 Journal of Nanomaterials

electrostatic interactions with negatively charged nucleicacids to form stable complexes [14 15] CS has been studiedfor more than a decade as a gene vector for pDNA oligonu-cleotides [16] and siRNA [17] In recent years low molecularweight (LMW) CS nanoparticles have shown great potentialin the applications of drug delivery and nonviral vector forgene delivery This is because compared with high molec-ular weight (HMW) chitosan LMW chitosan shows bettersolubility biocompatibility bioactivity biodegradability andeven less toxicity [18] To our knowledge until now there havebeen no studies investigating the use of LMW CS to deliverthe Dicer-substrate siRNA (DsiRNA) in vitro or in vivo

In 2005 some groups used slightly longer synthetic RNAsthat are substrates for Dicer and that are more potent thantraditional 21-mer siRNAs [19 20] These longer RNAs areprocessed by Dicer into 21-mer siRNAs in a predictablemanner [21] and the augmented potency seen with thisstrategy is thought to arise from the participation of Dicerin RNA-induced silencing complex formation Characteristi-cally these reagents are synthetic RNA duplexes that are 27bases in length and are referred to as DsiRNAs

In the current study LMWCSwas used and twomethodsof DsiRNA association were studied by simple complexationand adsorption of DsiRNA onto the surface of preformed CSnanospheres (CS NPs) To address the lack of informationregarding the interactions between DsiRNA and CS sinceDsiRNArsquos structure and size are relatively different fromthose of siRNA these systems were characterized in terms oftheir physical (particle size and distribution surface chargeand morphology) and biological features (cytotoxicity) Theefficiency of DsiRNA encapsulation binding and storagestability was also studied

2 Experimental

21 Materials Low molecular weight CS of molecularweight (MW) 190 kDa with a 75ndash85 degree of deacety-lation (DD) was obtained from Sigma-Aldrich (USA) andpentasodium tripolyphosphate (TPP) was obtained fromMerck (Germany) DsiRNA targeting the VEGF gene [51015840-rGrGrA rGrUrA rCrCrC rUrGrA rUrGrA rGrArU rCr-GrA rGrUA C-31015840 (sense strand) and 51015840-rGrUrA rCrUrCrGrArU rCrUrC rArUrC rArGrG rGrUrA rCrUrC rCrCrA-31015840 (antisense strand)] was purchased from Integrated DNATechnologies (IDT) USA Chinese hamster lung fibroblast(V79) cell lines and human colorectal adenocarcinoma cells(DLD-1) were obtained from American Type Culture Col-lection (ATCCManassas USA) Dulbeccorsquos Modified EaglersquosMedium (DMEM) fetal bovine serum (FBS) phosphatebuffered saline (PBS) and penicillin-streptomycin (pen-strep) were purchased from Gibco (USA) The alamarBluereagent was purchased from Invitrogen (USA) Acetic acidand other chemicals used were of analytical grade

22 Preparation of DsiRNA-CS Nanoparticles

221 Simple Complexation Four different concentrations ofCS solution (01 02 03 and 04 wv) were prepared

by diluting chitosan stock solution with deionized watercontaining 2 vv glacial acetic acid CS-DsiRNA complexeswere prepared by adding CS solution dropwise to an equalvolume of DsiRNA solution (15 120583gmL in deionized water)(Figure 1(a))Themixture was quicklymixed by inverting thereaction tube up and down for a few seconds and incubatingfor 30min at room temperature before further analysis

222 Ionic Gelation CS NPs were prepared via the ionicgelation method established by Calvo et al [22] with somemodifications CS solutions (01 02 03 and 04 wv)were prepared by dissolving CS in 2 vv glacial aceticacid TPP solution (1mgmL) was prepared by dissolvingin deionized water NPs were formed by adding 12mL ofTPP aqueous solution dropwise into 3mL CS solution (0403 02 and 01 wv) with constant magnetic stirring(MS MP8 Wise Stir Wertheim Germany) at 700 revolutionsper minute (rpm) for 30min at room temperature andincubated at room temperature for 30min before furtheranalysis NPs were collected by centrifugation (Optima L-100 XP Ultracentrifuge Beckman-Coulter California USA)at a speed of 35000 rpm at 10∘C for 30minThe supernatantswere discarded and the NPs were resuspended in filtered(Millex GP filter unit Millipore 025120583m) deionized waterThen DsiRNA was adsorbed onto the surface of CS NPsby adding 500120583L of DsiRNA solution (15 120583gmL) dropwiseinto 500120583L of CS NP suspension and quickly mixed byinverting the reaction tube up and down for a few seconds(Figure 1(b)) Finally the particles were incubated for 2 h atroom temperature before further analysis

223 ATR-FTIR Spectroscopic Analysis TheATR-FTIR spec-tra of naked DsiRNA CS TPP DsiRNA-CS NPscomplexesand blank CS NPs were recorded against the backgroundby using a universal ATR sampling assembly (Spectrum 100PerkinElmerWalthamMA USA) For each sample 16 scanswere obtained at a resolution of 4 cmminus1 in the range of 4000to 600 cmminus1

224 Determination of Particle Size and Zeta Potential Meanparticle size polydispersity index (PDI) and zeta potentialof CS NPs were measured on a ZS-90 Zetasizer (MalvernInstruments Worcestershire UK) that was based on photoncorrelation spectroscopy (PCS)

225 Morphological Analysis Morphological characteriza-tion of DsiRNA-CS NPs complexes was carried out by usingtransmission electron microscopy (TEM) Tecnai Spirit FEIEindhoven (The Netherlands) and atomic force microscopy(AFM) NTEGRA Prima device (NT-MDT Russia)

226 Determination ofDsiRNAEncapsulation Efficiency Theencapsulation efficiency () of DsiRNA complexed withCS or adsorbed onto CS NPs was measured by using UV-Vis spectrophotometer (Shimadzu 1800) at 260 nm from thefollowing formula

Journal of Nanomaterials 3

+

CS DsiRNAcomplexes CS-DsiRNA

CH2OH

n

H

H

OHlowast lowastO

NH3+

(a)

DsiRNA

CS TPP Nanoparticle CS-DsiRNA NPs

Na+Ominus

OminusNa+OminusNa+OminusNa+

OminusNa+

P P P+

CH2OH

n

H

H

OHlowast lowastO O O O

NH3+

(b)

Figure 1 Representation of CS nanoparticles prepared by simple complexation (a) and ionic gelation methods (b)

(Concentration of DsiRNA added minus Concentration of DsiRNA in supernatant)(Concentration of DsiRNA added)

times 100 (1)

227 Gel Retardation Assay The binding of DsiRNA to CSwas determined on a 4 wv agarose gel with SYBR Green(Invitrogen) A series of different CS to DsiRNAweight ratios(20120583L of sample containing 015120583g of DsiRNA) prepared bybothmethods were loaded into the wellsTheDsiRNA bandswere then visualized by using a real-timeUV transilluminator(Invitrogen USA)

228 Determination of Storage Stability In order to inves-tigate the storage stability of DsiRNA-CS NPscomplexes(prepared from 03 wv of CS) the mean diameter ofDsiRNA loaded CS NPscomplexes in deionized distilledwater was measured at 4∘ and 25∘C for a period of 15 days

23 In Vitro Release Study The release characteristics ofDsiRNA-CS NPscomplexes (CS concentration 03 wv)were studied in PBS (pH 72) Samples (4mL) were cen-trifuged at 35000 rpm for 30 min at 25∘C and deionizedwater was replaced with buffer solution (3mL) The mixturewas placed on a magnetic stirrer with a stirring speed of100 rpm at 37∘C for 15 days At different time intervalssamples were centrifuged at 35000 rpm for 30 min at 25∘CThen a whole volume of supernatant was removed foranalysis and replaced with an equivalent volume of freshbuffer solution The amount of released DsiRNA in thesupernatant was analyzed by a UV-visible spectrophotometer(Shimadzu 1800) at a wavelength of 260 nm

24 Cytotoxicity Study The V79 and DLD-1 cells (ATCCManassas USA) were cultured in DMEM and RPMI 1640

medium at cell density of 2 and 4 times 104 per well respectivelyBoth cell lines were supplemented with 10 FBS 1 pen-strep and maintained at 37∘C in a humidified 5 CO

2 95

air atmosphere At 24 and 48 h after incubation of DsiRNA-CS NPs or DsiRNA-CS complexes at 37∘C a final dilution of110 per cell volume of alamarBlue reagent was added to thetreated cells followed by a 4 h incubation prior to analysisTheabsorbance of each sample at 570 nm (A570) was measuredon a microplate reader (Varioskan Flash Thermo ScientificWaltham MA USA) Cell viability was determined by usingthe following equation

Cell viability () =119860570

of treated cells119860570

of control cellstimes 100 (2)

25 Statistical Analysis All the data were presented as meanplusmn standard deviation (SD) Data was analyzed with eitherthe independent Studentrsquos t-test or analysis of variance(ANOVA) followed by post hoc Tukeyrsquos analysis) by usingSPSS 190 For the independent t-test and ANOVA dif-ferences between tested groups were considered significantwhen 119875 lt 005

3 Results and Discussion

31 ATR-FTIR Spectroscopic Analysis ATR-FTIR spectra ofCS TPP naked DsiRNA CS-TPP NPs DsiRNA-CS NPs andthe DsiRNA-CS complexes are presented in Figure 2 Thespectrum of CS (Figure 2(a)) shows characteristic peaks at3430 cmminus1 (ndashOH stretching) 2882 cmminus1 (ndashCH stretching)


(a)

(b)

(c)

(d)

(e)

(f)1620 1350

34501610

1530

1540

16201530 1346

1200

895

3430 2882 1595 1324 1076

4000 3500 3000 2500 2000 1500 1000 550Wavenumber (cmminus1)

Tran

smitt

ance

(au

)

Figure 2 FTIR spectra containing 03 wv CS CS (a) TPP(b) DsiRNA (c) unloaded CS NPs (d) DsiRNA-CS NPs (e) andDsiRNA-CS complexes (f)

1595 cmminus1 (ndashNH2stretching) 1324 cmminus1 (CndashN stretching)

and 1076 cmminus1 (CndashOndashC stretching) In the spectrum of TPP(Figure 2(b)) a characteristic peak is observed at 895 cmminus1In the spectrum of DsiRNA (Figure 2(c)) characteristicPndashCH

3and PndashCH

2bending peaks are seen at 1346 cmminus1

and 1530 cmminus1 respectively Moreover P=O stretching wasobserved in the region of 1200 cmminus1 Another peak forDsiRNA is seen at 1620 cmminus1 The spectrum of unloadedCS NPs showed characteristic peaks for CS and TPP withslight shifting in the wavelengths and a decrease in intensity(Figure 2(d)) The peak for the NndashH bending vibration at1595 cmminus1 shifted to 1540 cmminus1 in the unloaded CS NPsafter addition of TPP In the spectrum for DsiRNA-CSNPs characteristic peaks for CS TPP and DsiRNA wereobserved which shows the presence of DsiRNA in CS NPs(Figure 2(e)) The ndashOH stretching was seen at 3450 cmminus1which indicates CSThe peaks obtained at 1610 and 1530 cmminus1confirmed the presence DsiRNA in the NPs In the spectrumof the DsiRNA-CS complex all characteristic peaks for CSand DsiRNA are seen (Figure 2(f)) Hence FTIR spectraconfirmed the successful preparation of theCSNPsDsiRNA-CS NPs and DsiRNA-CS complexes

32 Particle Size The mean particle size of CS NPs loadedwith DsiRNA prepared by either the simple complexation oradsorption method was significantly increased by increasingthe concentration of CS from 01 to 04 wv (ANOVATukeyrsquos post-hoc analysis 119875 lt 005) This was expecteddue to the decreased viscosity at the lower concentrationof CS which resulted in better solubility and propertiesconsistent with the polyelectrolyte type of materials Thisallowed for a more efficient interaction between negativelycharged DsiRNA and the cationic CS thus a smaller particlesize was produced [18]

The CS-DsiRNA complexes had a larger particle size ascompared to CS-DsiRNA NPs (independent Studentrsquos t-test119875 lt 005) as shown in Table 1 A larger particle size wasexpected due to the presence of intermolecular hydrogen

bonding (due to ndashOH groups) and higher intermolecularelectrostatic repulsion (due to ndashNH3+ groups) that existsalong the contour of CS [23] The smaller size of CS NPsadsorbed with DsiRNA could be due to quick gelling ofCS in contact with the polyanions (TPP) that relies onthe formation of inter- and intramolecular cross-linkagesmediated by the polyanions NPs were formed immediatelyuponmixing the TPP andCS solutions asmolecular linkageswere formed between the TPP phosphates and CS aminogroups This resulted in less electrostatic repulsion betweenthe amino groups of CS due to TPP neutralization [24] Inaddition DsiRNA adsorption onto the surface of CS NPs wasfound to have no significant effect on the particle size of CSNPs

33 Zeta Potential The comparative positive surface charge(zeta potential) of the DsiRNA loaded CS NPscomplexesincreased with increasing the concentration of CS at aconstant DsiRNA concentration (ANOVA Tukeyrsquos post-hocanalysis 119875 lt 005) as shown in Table 1 This increasewas due to the increase in the number of positive chargeswhich counteracts the negatively charged DsiRNA becausethe amount of DsiRNA was fixed A net positive chargefor the particles was desirable so as to prevent particleaggregation and promote electrostatic interaction with theoverall negative charge of the cell membrane [25]

The surface charge of CS NPs (unloaded) ranged approx-imately from +40 to +60mV (data not shown) by varyingthe CS concentration from 01 to 04 wv However theaddition of DsiRNA (15 120583gmL) to CS NPs showed a non-significant decrease in the zeta potentialThis finding differedfrom that of the siRNA-CS NPs which decreased in zetapotential after the adsorption process [18] This was thoughtto be due to the larger size of the DsiRNA duplex (27merMW16558 gmoL) as compared to the siRNAduplex (21merMW 13300 gmoL) In an equivalent volumetric solution ofDsiRNA and siRNA fewer units of DsiRNA than of siRNAwould be present in solution because of the larger size of theDsiRNA (27mer) As a result a fewer number of negativelycharged phosphate groups would be available in DsiRNAto compensate for the positive amine groups of the CSIn contrast more and smaller sized (21mer) siRNA units(and therefore more phosphate groups) would be present insolution to compensate for the amine groups of CS Thisobserved behaviour was in accordance to the pDNA [26]For this reason CS NPs loaded with DsiRNA showed a morepositive zeta potential than siRNA loaded CS NPs

34 Morphology The images of the CS NPs loaded withDsiRNA were obtained by TEM (Figure 3) and AFM(Figure 4) Figure 3(a) shows that unloadedCSNPs exhibiteda spherical structureTheDsiRNA-CSNPs also had the spher-ical morphology as depicted in Figures 3(b) 3(d) 3(f) and3(h) In contrast DsiRNA-CS complexes exhibited irregularshapes (Figures 3(c) 3(e) 3(g) and 3(i)) AFM micrographsof 03 wv CS also revealed a spherical morphology forDsiRNA-CS NPs (Figure 4(a)) and an irregular morphologyfor DsiRNA-CS complexes (Figure 4(b)) The heterogeneous


(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 3 TEM images ofNPscomplexes BlankCSNPs (03wvCS) (a)DsiRNA-CSNPs ((b) (d) (f) and (h)) andDsiRNA-CS complexes((c) (e) (g) and (i)) of 01 02 03 and 04 wv of CS respectively The magnification of the images were 43 kx ((a) (c) (d) and (f))60 kx ((e) and (g)) 20500x (b) 165 kx (h) and 135 kx (i)

(a) (b)

Figure 4 AFMmicrographs (containing 03 wv CS) DsiRNA-CS NPs (a) and DsiRNA-CS complexes (b) at 1 120583m scan size


Table 1 Particle size PDI and zeta potential of DsiRNA-CS NPscomplexes 119899 = 3

CS concentration ( 119908V)Simple complexation Adsorption

Particle size(nm) plusmn SD PDI plusmn SD Zeta potential

(mV) plusmn SDParticle size(nm) plusmn SD PDI plusmn SD Zeta potential

(mV) plusmn SD01 27033 plusmn 5614 060 plusmn 006 +5677 plusmn 764 21637 plusmn 4552 059 plusmn 006 +4050 plusmn 29502 40413 plusmn 1064 075 plusmn 017 +5710 plusmn 622 23100 plusmn 6878 052 plusmn 018 +4613 plusmn 42103 62467 plusmn 6490 069 plusmn 008 +6850 plusmn 187 25123 plusmn 3380 053 plusmn 010 +3727 plusmn 60704 73440 plusmn 7373 072 plusmn 019 +6923 plusmn 505 33650 plusmn 1138 074 plusmn 012 +6130 plusmn 491

0

20

40

60

80

100

01 02 03 04

Enca

psul

atio

n effi

cien

cy (

)

Concentration of CS ( wv)

DsiRNA-CS NPsDsiRNA-CS complexes

Figure 5 Encapsulation efficiency of DsiRNA-CS NPscomplexescontaining different CS concentrations 01 wv CS (a) 02 wvCS (b) 03 wv CS (c) and 04 wv CS (d)

(2) Naked DsiRNA(3) CS NPs(4) 01 wv CS

DsiRNA-CS (5) 02 wv CS NPs (6) 03 wv CS

(7) 04 wv CS (8) 01 wv CS DsiRNA-CS(9) 02 wv CS

(10) 03 wv CS complexes(11) 04 wv CS

330bp

100 bp

40bp

10bp

(1) DNA ladder (10bp)

1 2 3 4 5 6 7 8 9 10 11

Figure 6 Binding efficiency of DsiRNA loaded CS NPscomplexesas determined by 4 wv agarose gel electrophoresis

morphology of DsiRNA-CS complexes obtained in this studywas in agreement with previously reported studies on CScomplexes [27 28] The particle morphology is an importantfactor for the colloidal and chemical stability The resultsshowed that DsiRNA-CS NPs remained more stable at dif-ferent polymeric concentrations resulting in spherical mor-phology at different CS concentrations On the other handDsiRNA-CS complexes showed less stability at different poly-meric concentrations resulting in aggregation and irregularmorphology at different CS concentrationsThe difference inshapestability between nanospheres and complexes may bebest understood on the basis that the formation of CS NPsis governed not only by electrostatic interactions between theDsiRNA andCS but also by the interactions betweenTPP andCS The latter interaction was responsible for the controlledgelation of CS in a nanoparticulate form This controlledgelation and reticulation process could therefore explain whythe resulting nanoparticles were more spherical compactand stable than the simple complexes [26]

35 DsiRNA Encapsulation and Binding Efficiency A highDsiRNA encapsulation efficiency was obtained (100) forDsiRNA loaded CS NPscomplexes as measured by spec-trophotometry in Figure 5 For CS NPscomplexes loadedwith DsiRNA complete binding of DsiRNA to CS wasobserved (due to the absence of a trailing band) whichdemonstrates a strong interaction betweenCS andDsiRNAasshown in Figure 6 Overall these results showed thatDsiRNAwas efficiently and tightly associated to the nanoparticleshowever it was not irreversibly bound since it could bereleased upon the degradation of the polymeric matrix [2629]

36 Storage Stability The data corresponding to DsiRNA-CS NPscomplexes size progression over time is presented inFigure 7The rationale for conducting this study in deionizedwater was in the interest of obtaining information about theNPscomplexesrsquo stability in their suspending medium Thiscould help to avoid the need for extra procedures to stabilizethe NPscomplexes such as a lyophilization step Accord-ing to previous findings cross-linking reactions have beendescribed for improving the properties of the particulates[30] The results of this study support the previous findingthatDsiRNA-CS complexeswithout TPP tend to demonstratesome degree of size variation although no statistical signif-icance was seen at 4∘C and 25∘C as shown in Figures 7(a)and 7(b) respectively This was accompanied by an increase


0

200

400

600

800

1000

1200

1400

1600

0 5 10 15 20

Size

(nm

)

Time (days)


(a)

00

200

400

600

800

1000

1200

1400

1600

5 10 15 20Si

ze (n

m)

Time (days)


(b)

Figure 7 Stability study of DsiRNA-CS NPscomplexes at different temperature ranges At 4∘C (a) and 25∘C (b) 119899 = 3

0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16

Cum

ulat

ive D

siRN

A re

leas

e (

)

Time (days)


Figure 8 The release profile of DsiRNA loaded CS NPscomplexesat pH 72 119899 = 3

in PDI after 15 days of storage On the contrary DsiRNA-CSNPs containing TPP had decent stability at both temperaturesas shown in Figures 7(a) and 7(b) without a significant sizevariation during the experimental period of 15 days Theresults for the DsiRNA-CS NPs indicate that TPP acts as astabilizer possibly because of its cross-linking effect which

causes polymeric molecules to establish stronger interactionswith each other to form a more stable structure that is lessprone to aggregation as reported elsewhere [31]This suggeststhat DsiRNA-CS complexes behave as a metastable systemand thus they must be stored lyophilized and fresh aqueoussolutions should be prepared only when required HoweverDsiRNA-CS NPs were stable at both temperatures so theycould be stored at 4∘ and 25∘C without lyophilization

37 In Vitro Release of DsiRNA The in vitro release studyof DsiRNA from CS NPscomplexes was carried out inPBS to confirm the success of DsiRNA loading and tounderstand the release mechanism and kinetics of DsiRNAfrom CS NPscomplexes NPscomplexes prepared from aCS concentration of 03 wv were selected as they had thedesired characteristics (small nanosize net positive chargehigh entrapment and binding efficiency)The in vitro releaseprofiles of DsiRNA from the NPscomplexes were investi-gated for 15 days in PBS at pH of 72 Figure 8 illustrates thatthe release of DsiRNA could be divided into two stages basedon the release rate (the slope of the release profile) In the firststage the drug release pattern in DsiRNA-CSNPscomplexesshowed a rapid release in the first 4 days resulting in a 25and 19 cumulative release of DsiRNA in PBS (pH 72)respectivelyThe release ofDsiRNAat this stagemight involvethe diffusion of DsiRNA bound at the particle surface Inthe second stage DsiRNA was released at a constant rate(sustained release) from CS NPscomplexes for up to 15 daysAfter 15 days approximately 46 and 41 total recovered


0

20

40

60

80

100

120C

ell v

iabi

lity

()

125 250 500 1000

Simple complexationAdsorption

Concentration (120583gmL)cells

Untreated(05)

DsiRNA

(a)

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 10000



Untreated(05)

DsiRNA

(b)

Figure 9 Cytotoxicity effect of DsiRNA loaded CS NPscomplexes on V79 cells After 24 h (a) and 48 h (b) 119899 = 3

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 1000



Untreated(05)

DsiRNA

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 5001000



Untreated(05)

DsiRNA

(b)

Figure 10 Cytotoxicity effect of DsiRNA loaded CS NPscomplexes on DLD-1 cells After 24 h (a) and 48 h (b) 119899 = 3

amount of DsiRNA was released from CS NPscomplexesrespectively However no significant difference was observedin the DsiRNA cumulative amount released and in the releaserate from CS NPs and the complexes The nonsignificantdifference in the cumulative amount released and in therelease rate might be due to the fact that the dissolutionrate of the polymer near the surface was high therefore theamount of drug release from the surface would also be high[32]

38 Cytotoxicity Study To investigate the cytotoxic effectof DsiRNA-CS NPscomplexes on V79 and DLD-1 cells analamarBlue cell viability assay was performed In V79 cellsover 85 cell viability was observed for DsiRNA-CS NPsin comparison to untreated cells (Figure 9) Naked DsiRNAdid not show any loss of cell viability However 15ndash30 lossof cell viability was observed for CS-DsiRNA complexesA significant difference was observed in the cytotoxicity ofDsiRNA-CS NPscomplexes at 24 h and 48 h after incubation


(independent Studentrsquos t-test 119875 lt 005) The DsiRNA-CScomplexes exhibited more loss of cell viability than DsiRNA-CS NPs at 24 h after incubation (Figure 9(a)) The differencein the immediate cellular response between CS-DsiRNANPscomplexes might be due to the greater positive zetapotential of the CS-DsiRNA complexes which resulted in astronger interaction between CS-DsiRNA complexes and anegatively charged cell membrane leading to more loss ofcell viability Similarly DsiRNA-CS NPscomplexes showeda significant decrease in cell viability at 48 h after incubationas shown in Figure 9(b) (independent Studentrsquos t-test 119875 lt005) The cytotoxicity observed by both methods might bedue to a high positive zeta potential in CS NPscomplexesthat may interact with the negatively charged cell membraneMoreover DsiRNA-CS complexes showed greater loss of cellviability at 48 h after incubation thanDsiRNA-CSNPs whichmight be due to the greater positive zeta potential of thecomplexes that results in physiological stress to the cells

On DLD-1 cells DsiRNA-CS NPscomplexes exhibitedcytotoxic activity at 24 h and 48 h after incubation (Figures10(a) and 10(b)) Over a 15ndash35 and 10ndash28 loss in cellviability was observed for CS-DsiRNAcomplexes depend-ing on the concentration used An average of 7ndash8 loss incell viability was observed for DsiRNA in comparison tountreated cells No significant difference was observed in thecytotoxicity between DsiRNA CS NPscomplexes at 24 h and48 h after incubation in DLD-1 cells as depicted in Figures10(a) and 10(b) respectively Both NPs and complexes loadedwith DsiRNA showed a decrease in cell viability in DLD-1and V79 cells with DLD-1 cells having a slightly greater lossof cell viability However the difference in cell viability lossbetween DLD-1 and V79 cells by DsiRNA CS NPscomplexeswas not significantThe reason behind this loss of cell viabilityin DLD-1 cells might be due to CSrsquo ability to inhibit thegrowth of human cancer cells through an anti-angiogenicmechanism [33] Further investigation in different cell linesis still ongoing to address this effect

4 Conclusions

CS complexes and NPs loaded with DsiRNA were success-fully synthesized by simple complexation and ionic gelationmethods respectively FTIR analysis confirmed the success-ful encapsulation of DsiRNA ontowith CS NPscomplexesCS NPs obtained from the ionic gelation method showed itto be a better method as it produced smaller particles thatare expected to be more suitable for the efficient deliveryof DsiRNA to the target cells improved storage stability atdifferent temperatures and lesser toxicity in normal cellsIn contrast to that DsiRNA-CS complexes exhibited largerparticle size were more prone to aggregation and had highertoxicity for normal cells which may be due to their higherpositive zeta potential Nonetheless both methods exhibitedsustained release of DsiRNA over a period of time andhigh binding and high encapsulation (100) efficiencies Theresults of the present study therefore suggest that CS NPs(ionic gelation) could be used as a biocompatible nonviralgene delivery system This study also presents a platform for

further optimization studies of CS-based NPs such as stericstabilization and targeting

Conflict of Interests

The authors declare no conflict of interests with regard to thework

Acknowledgments

The authors gratefully acknowledge the financial support ofthis research by Exploratory Research Grant Scheme (ERGS)with Grant no ERGS12011SKKUKM0211 The authorswould like to thank all members of the Centre for DrugDelivery Research for their support

References

[1] H Katas N N S N Dzulkefli and S Sahudin ldquoSynthesis of anew potential conjugated TAT-peptide-chitosan nanoparticlescarrier via disulphide linkagerdquo Journal of Nanomaterials vol2012 Article ID 134607 7 pages 2012

[2] K Lundstrom ldquoLatest development in viral vectors for genetherapyrdquoTrends in Biotechnology vol 21 no 3 pp 117ndash122 2003

[3] S-D Li and L Huang ldquoNon-viral is superior to viral genedeliveryrdquo Journal of Controlled Release vol 123 no 3 pp 181ndash183 2007

[4] S H Kim H Mok J H Jeong S W Kim and T G ParkldquoComparative evaluation of target-specific GFP gene silencingefficiencies for antisense ODN synthetic siRNA and siRNAplasmid complexed with PEI-PEG-FOL conjugaterdquo Bioconju-gate Chemistry vol 17 no 1 pp 241ndash244 2006

[5] MHashida S TakemuraMNishikawa and Y Takakura ldquoTar-geted delivery of plasmid DNA complexed with galactosylatedpoly (L-lysine)rdquo Journal of Controlled Release vol 53 no 1ndash3pp 301ndash310 1998

[6] E Kleemann M Neu N Jekel et al ldquoNano-carriers forDNA delivery to the lung based upon a TAT-derived peptidecovalently coupled to PEG-PEIrdquo Journal of Controlled Releasevol 109 no 1ndash3 pp 299ndash316 2005

[7] J H Felgner R Kumar C N Sridhar et al ldquoEnhanced genedelivery and mechanism studies with a novel series of cationiclipid formulationsrdquo Journal of Biological Chemistry vol 269 no4 pp 2550ndash2561 1994

[8] F Liu and L Huang ldquoDevelopment of non-viral vectors forsystemic gene deliveryrdquo Journal of Controlled Release vol 78no 1ndash3 pp 259ndash266 2002

[9] H Katas Z Hussain and T C Ling ldquoChitosan nanoparticlesas a percutaneous drug delivery system for hydrocortisonerdquoJournal of Nanomaterials vol 2012 Article ID 372725 11 pages2012

[10] A G Luque-Alcaraz J Lizardi F M Goycoolea et al ldquoChar-acterization and antiproliferative activity of nobiletin-loadedchitosan nanoparticlesrdquo Journal of Nanomaterials vol 2012Article ID 265161 7 pages 2012

[11] M N Kumar R A Muzzarelli C Muzzarelli H Sashiwaand A J Domb ldquoChitosan chemistry and pharmaceuticalperspectivesrdquo Chemical Reviews vol 104 no 12 pp 6017ndash60842004


[12] K M Varum M M Myhr R J N Hjerde and O SmidsroslashdldquoIn vitro degradation rates of partially N-acetylated chitosansin human serumrdquo Carbohydrate Research vol 299 no 1-2 pp99ndash101 1997

[13] S B Rao and C P Sharma ldquoUse of chitosan as a biomaterialstudies on its safety and hemostatic potentialrdquo Journal ofBiomedical Materials Research vol 34 pp 21ndash28 1997

[14] M J Alonso and A Sanchez ldquoThe potential of chitosam inocular drug deliveryrdquo Journal of Pharmacy and Pharmacologyvol 55 no 11 pp 1451ndash1463 2003

[15] K Y Lee ldquoChitosan and its derivatives for gene deliveryrdquoMacromolecular Research vol 15 no 3 pp 195ndash201 2007

[16] S Gao J Chen L Dong Z Ding Y-H Yang and J ZhangldquoTargeting delivery of oligonucleotide and plasmid DNA tohepatocyte via galactosylated chitosan vectorrdquoEuropean Journalof Pharmaceutics and Biopharmaceutics vol 60 no 3 pp 327ndash334 2005

[17] H Katas andHOAlpar ldquoDevelopment and characterisation ofchitosannanoparticles for siRNAdeliveryrdquo Journal of ControlledRelease vol 115 no 2 pp 216ndash225 2006

[18] W Fan W Yan Z Xu and H Ni ldquoFormation mechanism ofmonodisperse low molecular weight chitosan nanoparticles byionic gelation techniquerdquo Colloids and Surfaces B vol 90 no 1pp 21ndash27 2012

[19] D Siolas C Lerner J Burchard et al ldquoSynthetic shRNAs aspotent RNAi triggersrdquo Nature Biotechnology vol 23 no 2 pp227ndash231 2005

[20] D-H Kim M A Behlke S D Rose M-S Chang S Choi andJ J Rossi ldquoSynthetic dsRNA Dicer substrates enhance RNAipotency and efficacyrdquo Nature Biotechnology vol 23 no 2 pp222ndash226 2005

[21] S D Rose D-H Kim M Amarzguioui et al ldquoFunctionalpolarity is introduced by Dicer processing of short substrateRNAsrdquo Nucleic Acids Research vol 33 no 13 pp 4140ndash41562005

[22] P Calvo C R Lopez J L Vila-Jato and M J AlonsoldquoNovel hydrophilic chitosan-polyethylene oxide nanoparticlesas protein carriersrdquo Journal of Applied Polymer Science vol 63no 1 pp 125ndash132 1997

[23] G Qun and W Ajun ldquoEffects of molecular weight degree ofacetylation and ionic strength on surface tension of chitosan indilute solutionrdquoCarbohydrate Polymers vol 64 no 1 pp 29ndash362006

[24] Q Gan T Wang C Cochrane and P McCarron ldquoModulationof surface charge particle size and morphological propertiesof chitosan-TPP nanoparticles intended for gene deliveryrdquoColloids and Surfaces B vol 44 no 2-3 pp 65ndash73 2005

[25] R M Schiffelers M C Woodle and P Scaria ldquoPharmaceuticalProspects for RNA Interferencerdquo Pharmaceutical Research vol21 no 1 pp 1ndash7 2004

[26] N Csaba M Koping-Hoggard and M J Alonso ldquoIoni-cally crosslinked chitosantripolyphosphate nanoparticles foroligonucleotide and plasmid DNA deliveryrdquo International Jour-nal of Pharmaceutics vol 382 no 1-2 pp 205ndash214 2009

[27] M Koping-Hoggard Y S Melrsquonikova K M Varum B Lind-man and P Artursson ldquoRelationship between the physicalshape and the efficiency of oligomeric chitosan as a genedelivery system in vitro and in vivordquo Journal of Gene Medicinevol 5 no 2 pp 130ndash141 2003

[28] P Erbacher S Zou T Bettinger A-M Steffan and J-S RemyldquoChitosan-based vectorDNA complexes for gene delivery

biophysical characteristics and transfection abilityrdquoPharmaceu-tical Research vol 15 no 9 pp 1332ndash1339 1998

[29] M E Martin and K G Rice ldquoPeptide-guided gene deliveryrdquoAAPS Journal vol 9 no 1 article 3 pp E18ndashE29 2007

[30] F-L Mi H-W Sung S-S Shyu C-C Su and C-K PengldquoSynthesis and characterization of biodegradable TPPgenipinco-crosslinked chitosan gel beadsrdquo Polymer vol 44 no 21 pp6521ndash6530 2003

[31] S Rodrigues A M R Costa and A GrenhaldquoChitosancarrageenan nanoparticles effect of cross-linkingwith tripolyphosphate and charge ratiosrdquo CarbohydratePolymers vol 89 no 1 pp 282ndash289 2012

[32] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B vol 84 no 1 pp 163ndash171 2011

[33] Y Xu Z Wen and Z Xu ldquoChitosan nanoparticles inhibit thegrowth of human hepatocellular carcinoma xenografts throughan antiangiogenic mechanismrdquoAnticancer Research vol 30 no12 pp 5103ndash5109 2009

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


electrostatic interactions with negatively charged nucleicacids to form stable complexes [14 15] CS has been studiedfor more than a decade as a gene vector for pDNA oligonu-cleotides [16] and siRNA [17] In recent years low molecularweight (LMW) CS nanoparticles have shown great potentialin the applications of drug delivery and nonviral vector forgene delivery This is because compared with high molec-ular weight (HMW) chitosan LMW chitosan shows bettersolubility biocompatibility bioactivity biodegradability andeven less toxicity [18] To our knowledge until now there havebeen no studies investigating the use of LMW CS to deliverthe Dicer-substrate siRNA (DsiRNA) in vitro or in vivo

In 2005 some groups used slightly longer synthetic RNAsthat are substrates for Dicer and that are more potent thantraditional 21-mer siRNAs [19 20] These longer RNAs areprocessed by Dicer into 21-mer siRNAs in a predictablemanner [21] and the augmented potency seen with thisstrategy is thought to arise from the participation of Dicerin RNA-induced silencing complex formation Characteristi-cally these reagents are synthetic RNA duplexes that are 27bases in length and are referred to as DsiRNAs

In the current study LMWCSwas used and twomethodsof DsiRNA association were studied by simple complexationand adsorption of DsiRNA onto the surface of preformed CSnanospheres (CS NPs) To address the lack of informationregarding the interactions between DsiRNA and CS sinceDsiRNArsquos structure and size are relatively different fromthose of siRNA these systems were characterized in terms oftheir physical (particle size and distribution surface chargeand morphology) and biological features (cytotoxicity) Theefficiency of DsiRNA encapsulation binding and storagestability was also studied

2 Experimental

21 Materials Low molecular weight CS of molecularweight (MW) 190 kDa with a 75ndash85 degree of deacety-lation (DD) was obtained from Sigma-Aldrich (USA) andpentasodium tripolyphosphate (TPP) was obtained fromMerck (Germany) DsiRNA targeting the VEGF gene [51015840-rGrGrA rGrUrA rCrCrC rUrGrA rUrGrA rGrArU rCr-GrA rGrUA C-31015840 (sense strand) and 51015840-rGrUrA rCrUrCrGrArU rCrUrC rArUrC rArGrG rGrUrA rCrUrC rCrCrA-31015840 (antisense strand)] was purchased from Integrated DNATechnologies (IDT) USA Chinese hamster lung fibroblast(V79) cell lines and human colorectal adenocarcinoma cells(DLD-1) were obtained from American Type Culture Col-lection (ATCCManassas USA) Dulbeccorsquos Modified EaglersquosMedium (DMEM) fetal bovine serum (FBS) phosphatebuffered saline (PBS) and penicillin-streptomycin (pen-strep) were purchased from Gibco (USA) The alamarBluereagent was purchased from Invitrogen (USA) Acetic acidand other chemicals used were of analytical grade

22 Preparation of DsiRNA-CS Nanoparticles

221 Simple Complexation Four different concentrations ofCS solution (01 02 03 and 04 wv) were prepared

by diluting chitosan stock solution with deionized watercontaining 2 vv glacial acetic acid CS-DsiRNA complexeswere prepared by adding CS solution dropwise to an equalvolume of DsiRNA solution (15 120583gmL in deionized water)(Figure 1(a))Themixture was quicklymixed by inverting thereaction tube up and down for a few seconds and incubatingfor 30min at room temperature before further analysis

222 Ionic Gelation CS NPs were prepared via the ionicgelation method established by Calvo et al [22] with somemodifications CS solutions (01 02 03 and 04 wv)were prepared by dissolving CS in 2 vv glacial aceticacid TPP solution (1mgmL) was prepared by dissolvingin deionized water NPs were formed by adding 12mL ofTPP aqueous solution dropwise into 3mL CS solution (0403 02 and 01 wv) with constant magnetic stirring(MS MP8 Wise Stir Wertheim Germany) at 700 revolutionsper minute (rpm) for 30min at room temperature andincubated at room temperature for 30min before furtheranalysis NPs were collected by centrifugation (Optima L-100 XP Ultracentrifuge Beckman-Coulter California USA)at a speed of 35000 rpm at 10∘C for 30minThe supernatantswere discarded and the NPs were resuspended in filtered(Millex GP filter unit Millipore 025120583m) deionized waterThen DsiRNA was adsorbed onto the surface of CS NPsby adding 500120583L of DsiRNA solution (15 120583gmL) dropwiseinto 500120583L of CS NP suspension and quickly mixed byinverting the reaction tube up and down for a few seconds(Figure 1(b)) Finally the particles were incubated for 2 h atroom temperature before further analysis

223 ATR-FTIR Spectroscopic Analysis TheATR-FTIR spec-tra of naked DsiRNA CS TPP DsiRNA-CS NPscomplexesand blank CS NPs were recorded against the backgroundby using a universal ATR sampling assembly (Spectrum 100PerkinElmerWalthamMA USA) For each sample 16 scanswere obtained at a resolution of 4 cmminus1 in the range of 4000to 600 cmminus1

224 Determination of Particle Size and Zeta Potential Meanparticle size polydispersity index (PDI) and zeta potentialof CS NPs were measured on a ZS-90 Zetasizer (MalvernInstruments Worcestershire UK) that was based on photoncorrelation spectroscopy (PCS)

225 Morphological Analysis Morphological characteriza-tion of DsiRNA-CS NPs complexes was carried out by usingtransmission electron microscopy (TEM) Tecnai Spirit FEIEindhoven (The Netherlands) and atomic force microscopy(AFM) NTEGRA Prima device (NT-MDT Russia)

226 Determination ofDsiRNAEncapsulation Efficiency Theencapsulation efficiency () of DsiRNA complexed withCS or adsorbed onto CS NPs was measured by using UV-Vis spectrophotometer (Shimadzu 1800) at 260 nm from thefollowing formula


+


CH2OH

n

H

H

OHlowast lowastO

NH3+

(a)

DsiRNA


Na+Ominus


OminusNa+

P P P+

CH2OH

n

H

H


NH3+

(b)



times 100 (1)






2 95









(a)

(b)

(c)

(d)

(e)

(f)1620 1350

34501610

1530

1540

16201530 1346

1200

895

3430 2882 1595 1324 1076


Tran

smitt

ance

(au

)




3and PndashCH










(a) (b) (c)

(d) (e) (f)

(g) (h) (i)


(a) (b)








0

20

40

60

80

100

01 02 03 04

Enca

psul

atio

n effi

cien

cy (

)








330bp

100 bp

40bp

10bp


1 2 3 4 5 6 7 8 9 10 11






0

200

400

600

800

1000

1200

1400

1600

0 5 10 15 20

Size

(nm

)

Time (days)


(a)

00

200

400

600

800

1000

1200

1400

1600

5 10 15 20Si

ze (n

m)

Time (days)


(b)


0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16

Cum

ulat

ive D

siRN

A re

leas

e (

)

Time (days)







0

20

40

60

80

100

120C

ell v

iabi

lity

()

125 250 500 1000



Untreated(05)

DsiRNA

(a)

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 10000



Untreated(05)

DsiRNA

(b)


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 1000



Untreated(05)

DsiRNA

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 5001000



Untreated(05)

DsiRNA

(b)







4 Conclusions





Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




+


CH2OH

n

H

H

OHlowast lowastO

NH3+

(a)

DsiRNA


Na+Ominus


OminusNa+

P P P+

CH2OH

n

H

H


NH3+

(b)



times 100 (1)






2 95









(a)

(b)

(c)

(d)

(e)

(f)1620 1350

34501610

1530

1540

16201530 1346

1200

895

3430 2882 1595 1324 1076


Tran

smitt

ance

(au

)




3and PndashCH










(a) (b) (c)

(d) (e) (f)

(g) (h) (i)


(a) (b)








0

20

40

60

80

100

01 02 03 04

Enca

psul

atio

n effi

cien

cy (

)








330bp

100 bp

40bp

10bp


1 2 3 4 5 6 7 8 9 10 11






0

200

400

600

800

1000

1200

1400

1600

0 5 10 15 20

Size

(nm

)

Time (days)


(a)

00

200

400

600

800

1000

1200

1400

1600

5 10 15 20Si

ze (n

m)

Time (days)


(b)


0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16

Cum

ulat

ive D

siRN

A re

leas

e (

)

Time (days)







0

20

40

60

80

100

120C

ell v

iabi

lity

()

125 250 500 1000



Untreated(05)

DsiRNA

(a)

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 10000



Untreated(05)

DsiRNA

(b)


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 1000



Untreated(05)

DsiRNA

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 5001000



Untreated(05)

DsiRNA

(b)







4 Conclusions





Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a)

(b)

(c)

(d)

(e)

(f)1620 1350

34501610

1530

1540

16201530 1346

1200

895

3430 2882 1595 1324 1076


Tran

smitt

ance

(au

)




3and PndashCH










(a) (b) (c)

(d) (e) (f)

(g) (h) (i)


(a) (b)








0

20

40

60

80

100

01 02 03 04

Enca

psul

atio

n effi

cien

cy (

)








330bp

100 bp

40bp

10bp


1 2 3 4 5 6 7 8 9 10 11






0

200

400

600

800

1000

1200

1400

1600

0 5 10 15 20

Size

(nm

)

Time (days)


(a)

00

200

400

600

800

1000

1200

1400

1600

5 10 15 20Si

ze (n

m)

Time (days)


(b)


0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16

Cum

ulat

ive D

siRN

A re

leas

e (

)

Time (days)







0

20

40

60

80

100

120C

ell v

iabi

lity

()

125 250 500 1000



Untreated(05)

DsiRNA

(a)

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 10000



Untreated(05)

DsiRNA

(b)


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 1000



Untreated(05)

DsiRNA

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 5001000



Untreated(05)

DsiRNA

(b)







4 Conclusions





Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b) (c)

(d) (e) (f)

(g) (h) (i)


(a) (b)








0

20

40

60

80

100

01 02 03 04

Enca

psul

atio

n effi

cien

cy (

)








330bp

100 bp

40bp

10bp


1 2 3 4 5 6 7 8 9 10 11






0

200

400

600

800

1000

1200

1400

1600

0 5 10 15 20

Size

(nm

)

Time (days)


(a)

00

200

400

600

800

1000

1200

1400

1600

5 10 15 20Si

ze (n

m)

Time (days)


(b)


0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16

Cum

ulat

ive D

siRN

A re

leas

e (

)

Time (days)







0

20

40

60

80

100

120C

ell v

iabi

lity

()

125 250 500 1000



Untreated(05)

DsiRNA

(a)

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 10000



Untreated(05)

DsiRNA

(b)


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 1000



Untreated(05)

DsiRNA

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 5001000



Untreated(05)

DsiRNA

(b)







4 Conclusions





Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









0

20

40

60

80

100

01 02 03 04

Enca

psul

atio

n effi

cien

cy (

)








330bp

100 bp

40bp

10bp


1 2 3 4 5 6 7 8 9 10 11






0

200

400

600

800

1000

1200

1400

1600

0 5 10 15 20

Size

(nm

)

Time (days)


(a)

00

200

400

600

800

1000

1200

1400

1600

5 10 15 20Si

ze (n

m)

Time (days)


(b)


0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16

Cum

ulat

ive D

siRN

A re

leas

e (

)

Time (days)







0

20

40

60

80

100

120C

ell v

iabi

lity

()

125 250 500 1000



Untreated(05)

DsiRNA

(a)

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 10000



Untreated(05)

DsiRNA

(b)


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 1000



Untreated(05)

DsiRNA

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 5001000



Untreated(05)

DsiRNA

(b)







4 Conclusions





Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

200

400

600

800

1000

1200

1400

1600

0 5 10 15 20

Size

(nm

)

Time (days)


(a)

00

200

400

600

800

1000

1200

1400

1600

5 10 15 20Si

ze (n

m)

Time (days)


(b)


0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16

Cum

ulat

ive D

siRN

A re

leas

e (

)

Time (days)







0

20

40

60

80

100

120C

ell v

iabi

lity

()

125 250 500 1000



Untreated(05)

DsiRNA

(a)

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 10000



Untreated(05)

DsiRNA

(b)


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 1000



Untreated(05)

DsiRNA

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 5001000



Untreated(05)

DsiRNA

(b)







4 Conclusions





Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

20

40

60

80

100

120C

ell v

iabi

lity

()

125 250 500 1000



Untreated(05)

DsiRNA

(a)

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 10000



Untreated(05)

DsiRNA

(b)


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 500 1000



Untreated(05)

DsiRNA

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

125 250 5001000



Untreated(05)

DsiRNA

(b)







4 Conclusions





Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






4 Conclusions





Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials


































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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